An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy.

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR+PD-1+ T cells. The anti-PD-1-GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1-GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

Localized inhibition of platelets and platelet derived growth factor by a matrix targeted glycan mimetic significantly attenuates liver fibrosis.

New therapeutic strategies are needed for the growing unmet clinical needs in liver disease and fibrosis. Platelet activation and PDGF activity are recognized as important therapeutic targets; however, no therapeutic approach has yet addressed these two upstream drivers of liver fibrosis. We therefore designed a matrix-targeting glycan therapeutic, SBR-294, to inhibit collagen-mediated platelet activation while also inhibiting PDGF activity. Herein we describe the synthesis and characterization of SBR-294 and demonstrate its potential therapeutic benefits in vitro and in vivo. In vitro SBR-294 was found to bind collagen (EC50 = 23 nM), thereby inhibiting platelet-collagen engagement (IC50 = 60 nM). Additionally, SBR-294 was found to bind all PDGF homodimeric isoforms and to inhibit PDGF-BB mediated hepatic stellate cell activation and proliferation. Translating these mechanisms in vivo, SBR-294 reduced fibrosis by up to 54% in the CCl4 mouse model (p = 0.0004), as measured by Sirius red histological analysis. Additional fibrosis measurements were also supportive of the therapeutic benefit in this model. These results support the therapeutic benefit of platelet and PDGF antagonism and warrant further investigation of SBR-294 as a potential treatment for liver fibrosis.

PrismPlus: a mouse line expressing distinct fluorophores in four different brain cell types.

To screen the complex central nervous system (CNS) injury responses, we created a quadruple-labelled 'PrismPlus' mouse line with a genetically encoded distinct fluorescent tag in oligodendrocytes, microglia, neurons, and astrocytes. Cx3cr1-gfp and Prism mice originally developed by Jung et al., 2000 and Dougherty et al., 2012, respectively, were cross-bred. First, we confirmed the presence of fluorophores in appropriate cell types in PrismPlus mice. PrismPlus mice were then used to examine the cellular responses to brain implanted micro-devices. We observed an increase in microglial response at earlier time points as compared to 4 weeks, a progressive astrocytic response, and fewer neurons at the vicinity of an implanted device. These results are similar to what has been described in literature using other rodent strains, previously attainable only through time-consuming and variable immunohistochemistry methods. Finally, we demonstrate the compatibility of PrismPlus brain tissue with CLARITY, an advanced tissue clearing technique, opening the door to future thick tissue imaging studies. This report confirms PrismPlus transgenic fluorescence and highlights the utility of these mice to study CNS injuries. The work herein seeks to establish a novel transgenic mouse line to improve experimental scope, consistency, and efficiency for CNS researchers.

Engineering and commercialization of human-device interfaces, from bone to brain.

Cutting edge developments in engineering of tissues, implants and devices allow for guidance and control of specific physiological structure-function relationships. Yet the engineering of functionally appropriate human-device interfaces represents an intractable challenge in the field. This leading opinion review outlines a set of current approaches as well as hurdles to design of interfaces that modulate transfer of information, i.a. forces, electrical potentials, chemical gradients and haptotactic paths, between endogenous and engineered body parts or tissues. The compendium is designed to bridge across currently separated disciplines by highlighting specific commonalities between seemingly disparate systems, e.g. musculoskeletal and nervous systems. We focus on specific examples from our own laboratories, demonstrating that the seemingly disparate musculoskeletal and nervous systems share common paradigms which can be harnessed to inspire innovative interface design solutions. Functional barrier interfaces that control molecular and biophysical traffic between tissue compartments of joints are addressed in an example of the knee. Furthermore, we describe the engineering of gradients for interfaces between endogenous and engineered tissues as well as between electrodes that physically and electrochemically couple the nervous and musculoskeletal systems. Finally, to promote translation of newly developed technologies into products, protocols, and treatments that benefit the patients who need them most, regulatory and technical challenges and opportunities are addressed on hand from an example of an implant cum delivery device that can be used to heal soft and hard tissues, from brain to bone.

Organic electrode coatings for next-generation neural interfaces.

Traditional neuronal interfaces utilize metallic electrodes which in recent years have reached a plateau in terms of the ability to provide safe stimulation at high resolution or rather with high densities of microelectrodes with improved spatial selectivity. To achieve higher resolution it has become clear that reducing the size of electrodes is required to enable higher electrode counts from the implant device. The limitations of interfacing electrodes including low charge injection limits, mechanical mismatch and foreign body response can be addressed through the use of organic electrode coatings which typically provide a softer, more roughened surface to enable both improved charge transfer and lower mechanical mismatch with neural tissue. Coating electrodes with conductive polymers or carbon nanotubes offers a substantial increase in charge transfer area compared to conventional platinum electrodes. These organic conductors provide safe electrical stimulation of tissue while avoiding undesirable chemical reactions and cell damage. However, the mechanical properties of conductive polymers are not ideal, as they are quite brittle. Hydrogel polymers present a versatile coating option for electrodes as they can be chemically modified to provide a soft and conductive scaffold. However, the in vivo chronic inflammatory response of these conductive hydrogels remains unknown. A more recent approach proposes tissue engineering the electrode interface through the use of encapsulated neurons within hydrogel coatings. This approach may provide a method for activating tissue at the cellular scale, however, several technological challenges must be addressed to demonstrate feasibility of this innovative idea. The review focuses on the various organic coatings which have been investigated to improve neural interface electrodes.

Intact histological characterization of brain-implanted microdevices and surrounding tissue.

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest. Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Chronic intracortical microelectrode arrays induce non-uniform, depth-related tissue responses.

OBJECTIVE: Brain-implanted microelectrode arrays show promise as future clinical devices. However, biological responses to various designs, compositions and locations of these implants have not been fully characterized, and may impact the long-term functionality of these devices. In order to improve our understanding of the tissue conditions at the interface of chronic brain-implanted microdevices, we proposed utilizing advanced histology and microscopy techniques to image implanted devices and surrounding tissue intact within brain slices. We then proposed utilizing these methods to examine whether depth within the cerebral cortex affected tissue conditions around implants. APPROACH: Histological data was collected from rodent brain slices containing intact, intracortical microdevices four weeks after implantation surgery. Thick tissue sections containing the chronic implants were processed with fluorescent antibody labels, and imaged in an optical clearing solution using laser confocal microscopy. MAIN RESULTS: Tissue surrounding microdevices exhibited two major depth-related phenomena: a non-uniform microglial coating along the device length and a dense mass of cells surrounding the implant in cerebral cortical layers I and II. Detailed views of the monocyte-derived immune cells improve our understanding of the close and complex association that immune cells have with chronic brain implants, and illuminated a possible relationship between cortical depth and the intensity of a chronic monocyte response around penetrating microdevices. The dense mass of cells contained vimentin, a protein not typically expressed highly in CNS cells, evidence that non-CNS cells likely descended down the face of the penetrating devices from the pial surface. SIGNIFICANCE: Image data of highly non-uniform and depth-dependent biological responses along a device provides novel insight into the complexity of the tissue response to penetrating brain-implanted microdevices. The presented work also demonstrates the value of in situ histological collection of brain implants for studying the complex tissue changes that occur, and the utility of pairing thick-tissue histology with appropriate optical clearing solutions.

Multimodal, longitudinal assessment of intracortical microstimulation.

The fundamental obstacle to neuroprostheses based on penetrating microstimulation is the tissue's response to the device insertion and to the application of the electrical stimulation. Our long-term goal is to develop multichannel microstimulation of central nervous tissue for clinical therapy. The overall objective of this research is to identify the optimal parameters for a chronically implanted microstimulation device. In particular, the work presented here focuses on the effects of repeated stimulation and the reactive tissue response on the efficacy of stimulation-driven behavior. To this end, psychophysical experiments were performed using multichannel cortical implants in the auditory cortex of rats. Further, we investigated the effect of the device-tissue interfacial quality on the psychophysical threshold. Here, we report the effects of cortical depth, days postimplant on the psychophysical threshold of auditory cortical microstimulation, along with correlated impedance spectral changes and post vivo histology. We expect that these data will further enable neuroprosthetic development.

In situ characterization of the brain-microdevice interface using device-capture histology.

Accurate assessment of brain-implantable microdevice bio-integration remains a formidable challenge. Prevailing histological methods require device extraction prior to tissue processing, often disrupting and removing the tissue of interest which had been surrounding the device. The Device-Capture Histology method, presented here, overcomes many limitations of the conventional Device-Explant Histology method, by collecting the device and surrounding tissue intact for subsequent labeling. With the implant remaining in situ, accurate and precise imaging of the morphologically preserved tissue at the brain/microdevice interface can then be collected and quantified. First, this article presents the Device-Capture Histology method for obtaining and processing the intact, undisturbed microdevice-tissue interface, and imaging using fluorescent labeling and confocal microscopy. Second, this article gives examples of how to quantify features found in the captured peridevice tissue. We also share histological data capturing (1) the impact of microdevice implantation on tissue, (2) the effects of an experimental anti-inflammatory coating, (3) a dense grouping of cell nuclei encapsulating a long-term implant, and (4) atypical oligodendrocyte organization neighboring a long term implant. Data sets collected using the Device-Capture Histology method are presented to demonstrate the significant advantages of processing the intact microdevice-tissue interface, and to underscore the utility of the method in understanding the effects of the brain-implantable microdevices on nearby tissue.

In vivo polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT) in rodent cerebral cortex.

Maintaining a reliable neural interface is a well-known challenge with implanted neural prostheses. Here we evaluate a method of forming an integrated neural interface through polymerization of PEDOT in vivo. Polymerization resulted in lower impedance and improved recording quality of local field potentials on implanted electrodes in the rat cerebral cortex. Histological analysis by optical microscopy confirmed successful integration of the PEDOT within tissue surrounding implanted electrodes. This technique offers a unique neural interfacing approach with potential to improve the long-term functionality of neural prostheses.

Errors in measuring blood gases in the intensive care unit: effect of delay in estimation.

Arterial blood gas measurement is subject to a number of potential sources of error. We investigated some of these in the intensive care unit (ICU). We audited samples for adequate volume and the presence of air and found that all samples were of adequate volume, but 40% contained bubbles or froth. We compared pulse oximeter estimations of oxygen saturation (SpO(2)) with laboratory estimates (SO(2)) from arterial blood samples, and found that there was less than a 5% chance of a difference of 5% or more. We audited the delay between sampling and processing and looked for errors arising as a result. We found that 4% of samples waited longer than 30 minutes to be analyzed in the laboratory, but that there was no correlation between delay and error in partial pressure of oxygen (PO(2)), carbon dioxide (PCO(2)), or SO(2). We performed a bench study to document the changes in PO(2) and PCO(2) over time with samples stored at room temperature and on ice. We found that samples in 1.5-mL PICO 70 syringes (Radiometer Medical A/S, Bronshoj, Denmark) were stable for PO(2) and SO(2) for up to 30 minutes either at room temperature or kept in iced water, and that changes after 60 minutes were small and unlikely to be clinically significant. PCO(2) showed a statistically significant increase after 20 minutes at room temperature, but the changes were not clinically significant.